Enzymatic activities in cell fractions of mycoplasmalike organisms purified from aster yellows-infected plants.

Mycoplasmalike organisms (MLOs), purified from aster yellows-infected plants were osmotically lysed, and the membranes were separated from the cytoplasmic fraction through differential centrifugation. Electron microscopic examinations of sections of the purified MLOs and the isolated membranes showed pleomorphic bodies and unit membranous empty vesicles, respectively. Cell fractions were tested for NADH oxidase, NADPH oxidase, ATPase, RNase, DNase, and p-nitrophenyl phosphatase activity. NADH oxidase and ATPase were confined to the membrane fraction and NADPH oxidase to the cytoplasmic fraction of the MLOs. para-Nitrophenyl phosphatase, RNase, and DNase activities were detected in both membrane and cytoplasmic fractions, but p-nitrophenyl phosphatase and RNase appeared to be associated with membranes and DNase with the cytoplasmic fraction. Glucose-6-phosphate dehydrogenase was found in the cytoplasmic fraction of the MLO cells. Our findings on the distribution of enzymes in MLO cells and cell fractions are the first basic documentation on nonhelical, nonculturable microbes parasitic to plants.     

Studies of the mycoplasma-like organism (MLO) in spinach leaves affected by the aster yellows disease

Summary Mycoplasma-like organisms (MLO) were found in the phloem of spinach (Spinacia oleracea L.) leaves infected with the agent of aster yellows disease by means of the leafhopperMacrosteles fascifrons Stål. The MLOs occurred mainly in mature sieve elements but were recorded in occasional phloem parenchyma cells as well. The MLO showed the typical features of this organism. The majority were ovoid or spherical, some were irregular in form or elongated. The larger bodies were commonly accompanied by small bodies which appeared to originate from the larger by budding. Profiles suggesting binary fission and filamentous forms containing ovoid condensations of cytoplasm were present. The bounding membrane showed the typical trilaminate structure, and DNA-like fibrils were discernible in those MLOs that had an electron lucent central region. In the denser bodies the fibrils were obscured. The MLO ribosomes were distinctly smaller than those in the host cytoplasm. The MLOs were degenerating in phloem cells that were disorganizing and collapsing in response to the infection. Structures in host cells that may be confused with MLO are described.This work was supported by the National Science Foundation grant GB-35228 to K. E and by Hatch and California Statewide Critical Applied Research Funds to the Departmem of Cell Physiology, University of California, Berkeley, California. The authors thank ProfessorJulius H.Freitag for providing the original strains of the aster yellows agent.     

Identification of aster yellows phytoplasma in garlic and green onion by PCR-based methods

In the summer of 1999, typical yellows-type symptoms were observed on garlic and green onion plants in a number of gardens and plots around Edmonton, Alberta, Canada. DNA was extracted from leaf tissues of evidently healthy and infected plants. DNA amplifications were conducted on these samples, using two primer pairs, R16F2n/R2 and R16(1)F1/R1, derived from phytoplasma rDNA sequences. DNA samples of aster yellows (AY), lime witches'-broom (LWB) and potato witches'-broom (PWB) phytoplasmas served as controls and were used to determine group relatedness. In a direct polymerase chain reaction (PCR) assay, DNA amplification with universal primer pair R16F2n/R2 gave the expected amplified products of 1.2 kb. Dilution (1/40) of each of the latter products were used as template and nested with specific primer pair R16(1)F1/R1. An expected PCR product of 1.1 kb was obtained from each phytoplasma-infected garlic and green onion samples, LWB and AY phytoplasmas but not from PWB phytoplasma. An aliquot from each amplification product (1.2 kb) with universal primers was subjected to PCR-based restriction fragment length polymorphism (RFLP) to identify phytoplasma isolates, using four restriction endonucleases (AluI, KpnI, MseI and RsaI). DNA amplification with specific primer pair R16(1)F1/R1 and RFLP analysis indicated the presence of AY phytoplasma in the infected garlic and green onion samples. These results suggest that AY phytoplasma in garlic and green onion samples belong to the subgroup 16Sr1-A.     

Identification and grouping of mycoplasmalike organisms associated with grapevine yellows and clover phyllody diseases based on immunological and molecular analyses.

Immunofluorescent staining, dot blot hybridization, PCR, random amplified polymorphic DNA (RAPD) markers, and restriction fragment length polymorphism wee used to study the genetic relatedness among mycoplasmalike organisms (MLOs) associated with several geographically diverse grapevine yellows diseases (CA1, CH1, SA1, and SA2 from Bologna, Italy; GYU from Udine, Italy; GYR from Rome, Italy; and GYG from Germany). The relationship between these and MLOs associated with clover phyllody diseases in Italy (CPhB and CPhC) and Canada (CPhCa) was also examined. Two monoclonal antibodies reacted with MLOs of GYU-, CPhB-, and CPhC-infected periwinkles. Dot blot hybridization with two cloned GYU DNA fragments, GYD-1 and GYD-2 inserts, showed that both hybridized with DNAs of GYU-, CPhB-, and CPhC-infected periwinkles but not with those of GYR and CPhCa. In addition, GYD-1 insert hybridized with DNAs of CA1, CH1, SA1, SA2, and GYG. Three primer pairs were developed in PCR experiments for this study. By using primer set GYD2P1F and GYD2P1R, a 600-bp DNA fragment was amplified only when DNAs from GYU-, CPhB-, and CPhC-infected plants were used as templates. With the primer pair GYD2P1F and GYD2P2R, a 550-bp DNA fragment was amplified from GYU, CPhB, CPhC, and GYG. The primer pair GYD1P1F and GYD1P2R, on the other hand, could amplify all isolates, although the patterns of PCR products were not identical for all isolates.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cloned riboprobe for detection of a mycoplasmalike organism

A [32P]-labeled single stranded-RNA probe (riboprobe) was constructed with plasmid vector pSP64 and used to detect and specifically identify an uncultured pathogenic mycoplasmalike organism in infected host. The riboprobe was more sensitive and reliable than complementary double stranded-DNA probe in detection of western X mycoplasmalike organism. When concentration of a double stranded-DNA probe was increased, nonspecific hybridization signal was observed with nucleic acid from healthy plants and from plants infected by other mycoplasmalike organisms. In contrast, sensitivity of detection with the complementary riboprobe was increased at elevated probe concentrations without nonspecific hybridization.     

Response of mycorrhizal periwinkle plants to aster yellows phytoplasma infection

The objective of our research was to assess if arbuscular mycorrhizal (AM) fungal colonization can modify the effect of infection by two aster yellows phytoplasma strains (AY1, AYSim) in Catharanthus roseus plants. Both phytoplasma strains had a negative effect on the root fresh weight, but they differed in symptoms appearance and in their influence on photosynthetic and transpiration rates of the periwinkle plants. AM plants showed significantly reduced shoot fresh weight, while the transpiration rate was significantly increased. AM fungal colonization significantly affected shoot height and fresh weight of the plants infected by each phytoplasma strains as well as the root system of plants infected with the more aggressive AYSim phytoplasma strain. Double inoculation did not reduce the negative effects induced with phytoplasma alone on the photosynthetic activity of phytoplasma-infected plants.     

Spatial pattern analysis of the incidence of aster yellows disease in lettuce

The degree of aggregation of lettuce plants infected by aster yellows phytoplasma (AYP) was investigated in 12 fields from three experiments. Position of diseased and healthy plants was mapped in a 6–9×12-m section of each field; for most analyses, fields were divided into 10-plant quadrats. Mean disease incidence (p) ranged from 0.01 to 0.30. The frequency of diseased plants was described by the beta-binomial distribution, with an index of aggregation (θ) ranging from 0 to 0.17, positively correlated withp, and generally increasing over time within a field. Distance-class analysis revealed a core-cluster size of only a few plants. However, spatial autocorrelations ofp between quadrats were not significant, indicating that the scale of spatial pattern was small, generally less than 10 plants. An overall measure of aggregation was given by the slope parameter of the binary form of the power law, in which the log of the calculated variance is regressed on the log of the theoretical variance for a binomial distribution. The slope was 1.18 and significantly different from 1. Results for this “simple-interest” disease are interpreted in relation to the persistent transmission of AYP by its aster leafhopper vector.     

Evolutionary relationships of a plant-pathogenic mycoplasmalike organism and Acholeplasma laidlawii deduced from two ribosomal protein gene sequences.

The families within the class Mollicutes are distinguished by their morphologies, nutritional requirements, and abilities to metabolize certain compounds. Biosystematic classification of the plant-pathogenic mycoplasmalike organisms (MLOs) has been difficult because these organisms have not been cultured in vitro, and hence their nutritional requirements have not been determined nor have physiological characterizations been possible. To investigate the evolutionary relationship of the MLOs to other members of the class Mollicutes, a segment of a ribosomal protein operon was cloned and sequenced from an aster yellows-type MLO which is pathogenic for members of the genus Oenothera and from Acholeplasma laidlawii. The deduced amino acid sequence data from the rpl22 and rps3 genes indicate that the MLOs are more closely related to A. laidlawii than to animal mycoplasmas, confirming previous results from 16S rRNA sequence comparisons. This conclusion is also supported by the finding that the UGA codon is not read as a tryptophan codon in the MLO and A. laidlawii, in contrast to its usage in Mycoplasma capricolum.     

Occurrence and identification of aster yellows related phytoplasma in Gladiolus in Poland

In 1996–1998 on Gladiolus plants cultivated in Poland severe symptoms were observed. The symptoms included chlorosis of the youngest leaves, yellowing and malformation of flower spices, flower discoloration and virescence. The affected corms kept in cold storage developed premature multiple sprouts weak and pale in color. Their root formation was strongly inhibited. Electron microscopy examination of the ultra-thin sections of the leaves and roots of diseased plants showed necrosis and collapsing of sieve tubes and companion cells, reduction of phloem and xylem strands as well as decrease of the number and diameter of xylem vessels. Numerous polymorphic bodies were observed in the phloem and parenchyma cells of affected gladioli. PCR amplification using universal phytoplasma primers rU3 and fU5 directed to ribosomal sequences and RFLP analysis of the amplified rDNA were used to identify the phytoplasma causing yellow disease in Poland. Specific product of about 880 bp was obtained, providing evidence of phytoplasma infection. RFLP analysis of the PCR product done with restriction enzyme AluI showed that the diseased gladioli were infected by phytoplasma very similar or identical with American aster yellows phytoplasma.     

Molecular and microscopical detection of aster yellows phytoplasma associated with infected parsnip

Typical phytoplasma yellows symptoms were observed in parsnip (Pastinaca sativa L.) plants grown around Edmonton, Alberta, Canada. Examination of ultrathin sections of leaf midribs by electron microscopy revealed numerous phytoplasma bodies localized in the phloem cells. DNA extracted from the infected leaves was amplified with a 16S rDNA universal primer pair P1/P6 giving the expected PCR product of 1.5 kb. The phytoplasma was confirmed as a member of the aster yellows (AY) group by amplification with the specific primer pair R16(1)/F1/R1 that was designed on the basis of AY phytoplasma 16S rDNA sequences. In the nested PCR assays, the expected DNA fragment of 1.1 kb was amplified with this specific primer set. Similar restriction patterns were found for the 1.1 kb PCR products of the phytoplasma isolated from parsnip and an AY phytoplasma control after digestion with restriction endonucleases AluI, HhaI, KpnI and RsaI. This is the first reported observation of aster yellows in parsnip in Canada.     

Banded filaments associated with the aster yellows MLO in Vinca rosea L

The ultrastructure of an aster yellows mycoplasma-like organism was studied in the phloem of periwinkle (Vinca rosea L.) plants. Banded filaments were observed in association with mycoplasma-like organisms of characteristic morphology. The filaments were variable in length, from 50-100 nm in width, and displayed a regular periodic banding of alternating electron-dense and electron-lucent structures.     

The genome size of a plant-pathogenic mycoplasmalike organism resembles those of animal mycoplasmas.

The genome size of a mycoplasmalike organism was determined by comparing fluorescence intensities of restriction fragments. Its genome size was similar to that of Mycoplasma gallisepticum and much smaller than that of Acholeplasma laidlawii. Although the genome size is "mycoplasmalike," other molecular data indicate a closer evolutionary relationship to A. laidlawii.     

Detection and molecular characterization of an aster yellows phytoplasma in poker statice and Queen Anne's lace in Alberta, Canada

Queen Anne's lace and poker statice plants were found with a yellows-type disease with typical phytoplasma symptoms in an experimental farm near Brooks, Alberta in 1996. Phytoplasma bodies were detected by transmission electron microscopy in phloem cells of symptomatic plants, but not in healthy plants. The presence of a phytoplasma was confirmed by analysis with the polymerase chain reaction. Using a pair of universal primer sequences derived from phytoplasma 16S rRNA, an amplified product of the expected size (1.2 kb) was observed in samples from infected plants, but not in asymptomatic plants. Sequence analysis of the PCR products from the 16S/23S rDNA intergenic spacer region indicated that the two phytoplasma isolates in Queen Anne's lace and poker statice are genetically closely related to the western aster yellows phytoplasma.     

Detection of aster yellows phytoplasma in false flax based on PCR and RFLP

False flax (Camelina sativa L.) plants were found to be infected with a yellows-type disease caused by a phytoplasma in experimental plots at the Edmonton Research station. Alberta, Canada. Typical phytoplasmas were detected in the phloem cells in ultrathin sections from leaf midrib tissues examined by electron microscopy. These observations were supported by polymerase chain reaction (PCR) using two primer pairs, R16 F2n/R2 and R16(1)F1/R1, derived from phytoplasma rDNA sequences. Aster yellows (AY) and potato witches'-broom (PWB) phytoplasma DNA samples served as controls and were used to study group relatedness. In a direct PCR assay, DNA amplification with universal primer pair R16F2n/R2 gave the expected PCR products of 1.2 kb. Based on a nested-PCR assay using the latter PCR products as templates, and a specific primer pair, R16(1)F1/R1, designed on the basis of AY phytoplasma rDNA sequences, a PCR product of 1.1 kb was obtained from each phytoplasma-infected false flax and AY sample, but not from PWB phytoplasma and healthy controls. DNA amplification with specific primer pair R16(1)F1/R1 and restriction fragment length polymorphism indicated the presence of AY phytoplasma in the infected false flax sample. This is the first reported characterization of AY phytoplasma in false flax.     

Multigene analysis for differentiation of aster yellows phytoplasmas infecting carrots in Serbia

During a survey of large carrot fields in Serbia, plants showing leaf reddening and/or yellowing, adventitious shoot production and reduction in taproot size and quality were observed in a low percentage of plants. To verify phytoplasma association with the described symptoms and to carry out pathogen differentiation, PCR assays followed by restriction fragment length polymorphism (RFLP) analyses and/or sequencing of phytoplasma 16Sr DNA and ribosomal protein genes l22 and s3 , tuf , putative aa kinase plus ribosomal recycling factor genes and DNA helicase gene were carried out. Phytoplasmas belonging to 16SrI-A and 16SrI-B ribosomal subgroups and to rpI-A and rpI-B ribosomal protein subgroups, respectively, were identified by RFLP analyses in 13 of 15 symptomatic plants tested. No amplification was obtained with non-symptomatic carrot samples. The identification was confirmed by sequence analyses of the phytoplasma genes studied. In two carrot samples, presence of interoperon sequence heterogeneity was detected and phytoplasma strains were identified as belonging to 16SrI group but were not assigned to any 16S rRNA or ribosomal protein subgroup. This research allowed the first molecular identification of phytoplasmas infecting carrot in Serbia using several molecular markers, and it indicates that under field conditions in non-epidemic outbreaks a certain amount of genetic mutation may occur in conserved genes of these prokaryotes.     

Nucleotide sequence of beet western yellows virus RNA.

The nucleotide sequence of the genomic RNA (5641 nt) of beet western yellow virus (BWYV) isolated from lettuce has been determined and its genetic organization deduced. The sequence of the 3'terminal 2208 nt of RNA of a second BWYV isolate, obtained from sugarbeet, was also determined and was found to be very similar but not identical to that of the lettuce isolate. The complete sequence of BWYV RNA contains six long open reading frames (ORFs). A cluster of three of these ORFs, including the coat protein cistron, display extensive amino acid sequence homology with corresponding ORFs of a second luteovirus, the PAV isolate of barley yellow dwarf virus (BYDV) (1,2). The ORF corresponding to the putative viral RNA-dependant RNA polymerase, on the other hand, resembles that of southern bean mosaic virus. There is circumstantial evidence that expression of the BWYV RNA polymerase ORF may involve a translational frameshift mechanism. The ORF immediately following the coat protein cistron may be translated by in-frame readthrough of the coat protein cistron amber termination codon. Similar mechanisms have been proposed for expression of the corresponding ORFs of BYDV(PAV) (1).